In this proposal we plan to examine the relationship between the effects of estrogen withdrawal on bone and the ability of bone cells to respond to the cytokines IL-1 and TNF. We will first determine if estrogen withdrawal can cause the loss of bone in mice that are deficient in either the IL-1 type 1 receptor (IL-1R1 minus/minus), TNF receptor 1 (TNFR1 minus/minus) or both. I will determine that role that responses to the cytokines IL-1 and TNF have on the ability of resorption stimuli to increase osteoclast-like cell (OCL) formation rates in vitro. Spleen cells, a source of osteoclast precursor cells and primary osteoblast- enriched cells, which contain supporting cells for osteoclast formation will be co-cultured with resorption stimuli. By selectively using combinations of wild type and cytokine deficient spleen cells and osteoblastic cells, e will determine the importance of IL-1R1 and TNFR1 in the ability of a variety of resorption stimuli to increase OCL formation rates in vitro. In addition, we should be able to determine on which cells IL-1 and TNF must act for OCL formation to be increased by each stimulus. We will determine the ability of cell cultures from ovariectomized (OVX) mice to increase in vitro OCL formation compared to cell cultures from sham-operated (SHAM) or ovariectomized and estrogen treated (OVX plus E) mice. OCL formation will be studied in whole bone marrow cultures that contain both osteoclast precursors and support cells and in spleen cell-osteoblast co- cultures. By selectively examining cells from wild type, IL-1R1- and TNFR1- mice that have been either SHAM, OVX or OVX or OVX plus E treated, we will determine the importance of each cytokine receptor in the ability of estrogen withdrawal to stimulate osteoclast formation. We will also determine on which cells (osteoclast precursor or osteoblastic support cell) the cytokine receptors need to be present for estrogen withdrawal to stimulate osteoclast formation rates in vitro. We will further characterize the factors in marrow supernatants (Msups) that regulate IL-1 activity and are altered by OVX treatment. We will determine the IL-1 bioactivity of Msups in a variety of assays. We will also determine the IL-1 binding proteins in Msups and the effects of Msups on IL-1 binding and receptor number in bone and bone cells.